Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
  • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
  • C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
  • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

Definitions

• This invention relates to a novel compound N-[4-(3-aminopropyl)aminobutyl]-2-( ⁇ -guanidino-fatty-acid-amido)-2-substituted-ethanamide which is useful as a carcinostatic substance and is represented by the general formula ##STR4## wherein Y represents --CH 2 --CH 2 --, --CH ⁇ CH-- or ##STR5## R represents a hydrogen atom, an alkyl group of 1 to 4 carbon atoms which may have a hydroxyl group as substituent, or a benzyl group, and n is an integer of from 1 to 8, provided that when Y is ##STR6## and n is 4, R represents the groups other than the hydrogen atom; a salt thereof and a process for the preparation thereof.
• the chemical structure of spergualin is represented by the formula ##STR7##
• the configuration at the position 15 is S, while that at the position 11 is yet to be determined [Journal of Antibiotics, Vol. 34, 1622 (1981)].
• the compound of this formula is synthesized by the condensation of the acid amide and glyoxylylspermidine (Umezawa et al., U.S. patent application Ser. Nos. 375,916, now abandoned, and 375,950, now U.S. Pat. No. 4,430,346, filed May 5 and May 7, 1982, respectively).
• the resulting epimeric compound is resolved into natural (-)-spergualin and non-natural (+)-spergualin [Journal of Antibiotics, Vol. 34, 1625 (1981)].
• This invention is based on the discovery that the compounds represented by the general formula I exhibit an excellent antitumor activity in animals. Those wherein R is a group other than a hydrogen atom, have excellent stability relative to those wherein R is a hydrogen atom.
• the compounds of general formula I in which R is a hydrogen atom are produced by the condensation of an ⁇ -guanidino fatty acid amide of the general formula ##STR8## wherein Y and n are as defined above, and N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide of the formula ##STR9##
• R of the general formula I is a group other than hydrogen
• the compounds are synthesized by reacting the compound obtained by the above condensation or spergualin obtained from the microbial culture filtrate, in which the amino and imino groups may be protected, with an aliphatic mono- or dialcohol of 1 to 4 carbon atoms, a diazoparaffin of 1 to 4 carbon atoms, or benzyl alcohol, and removing the amino or imino protective groups when present.
• the compounds of this invention are used generally in the form of a pharmacologically acceptable acid addition salt.
• acid addition salts mention may be made of salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and boric acid, and organic acids such as acetic acid, citric acid, tartaric acid and glutaric acid.
• the compounds of this invention represented by the general formula ##STR10## wherein Y, R and n are as defined above, each has an asymmetric carbon at the position 11 and, hence, exists in the form of epimers with respect to carbon 11, that is, in the form of levorotatory epimer [hereinafter referred to as (-)] and in the form of dextrorotatory epimer [hereinafter referred to as (+)]. Unless specifically indicated, the present compound is a mixture (approximately 1:1) of a pair of epimers [if necessary, referred to as ( ⁇ )]. ##STR11## carbon at position 15 and, hence, there is one epimer having S-configuration at the position 15 and the other epimer having R-configuration at the position 15. Unless specifically indicated, the present compound is a mixture (approximately 1:1) of (S)-epimer and (R)-epimer.
• the chemical stability of the present compound was evaluated by determining the retention (%) after heating at 60° C. for 4 hours.
• Table 5 are shown the test results together with those for spergualin as reference.
• the retention was determined by means of high performance liquid chromatography (HPLC).
• HPLC high performance liquid chromatography
• the column was packed with Nucleosil® 5C18.
• the solvent used for spergaulin was a mixture (6:94) of acetonitrile--0.01M sodium pentanesulfonate+0.01M Na 2 HPO 4 (pH 3), while that for the compounds of this invention was a mixture of acetonitrile--0.005M sodium pentanesulfonate+0.01M Na 2 HPO 4 (pH 3).
• the mixing ratio in the latter case was varied for each particular compound. For instance, a mixing ratio of 10:90 was used for Compound No. 9, while that of 7:93 for Compound No. 22.
• All of the compounds of this invention possess a strong growth-inhibitory effect on cancer cells in vitro and a life-prolonging effect on mice bearing implanted cancer cells, as shown below.
• mice were implanted with 10 5 mouse leukemia L-1210 cells.
• the ascites collected aseptically from the mice after 4 days of feeding was washed three times with physiological saline to obtain L-1210 cells, which was then suspended in RPMI 1640 culture medium [G. E. Moore, Journal of the American Medical Association, Vol. 199, 519 (1967); H. J. Morton, In Vitro, Vol. 6, 89 (1970)] to which 10% fetal calf serum and 5 ⁇ M 2-mercaptoethanol had been added, and the resulting suspension was diluted to 5 ⁇ 10 4 L-1210 cells per 0.9 ml.
• the 50% growth inhibitory concentration was calculated from the values of growth inhibition at varied sample concentrations.
• the results of tests on typical compounds of this invention for the growth inhibition of L-1210 cells were as shown in Table 6.
• the therapeutic effect of typical compounds of this invention on mouse leukemia L-1210 were as shown in Table 7.
• the compounds of this invention represented by the formula I have antitumor activity in animals. They exhibit an excellent growth-inhibitory activity against mouse leukemia L-1210 cells.
• R is a hydrogen atom, an alkyl group of 1 to 4 carbon atoms which may have a hydroxyl group as substituent, or a benzyl group, and n is an integer of from 1 to 6, provided that when Y is ##STR17## R represents the groups other than the hydrogen atom.
• R is a hydrogen atom or an alkyl group of one or two, preferably one, carbon atom, which may have a hydroxyl group as substituents, and n is 4 or 6.
• R is a hydrogen atom or an alkyl group of one or two, preferably one, carbon atom, which may have a hydroxyl group as substituents, and n is 4 or 6.
• R is a methyl group (an alkyl group of one carbon atom) are excellent also in chemical stability, compound Nos. 9, 19 and 22 being most desirable.
• the invention includes the method for inhibiting tumor growth with a compound Formula I.
• the compound is administered systemically, preferably by parenteral injection and on a repetitive dosage regimen, to a tumor-bearing mammal in a non-toxic tumor-inhibitory effective amount.
• acetone and dimethylformamide are suitable, but the condensation is generally conducted in the presence of small amounts of water without using organic solvents.
• the amount of water to be used should be the least possible for dissolving both compounds. In practice, it is used in an amount in the range of from 2 to 60, preferably from 4 to 40, moles for 1 mole of the guanidino fatty acid amide of the formula II. Since these compounds of formulas II and III are usually treated in the form of acid addition salt, it is not necessary to add an acid. However, in view of the yield of condensate, it is preferable to use an acid catalyst.
• Suitable acid catalysts include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and boric acid and organic acids such as acetic acid, citric acid, tartaric acid, succinic acid, glutaric acid and adipic acid.
• a dicarboxylic acid such as glutaric acid is preferred.
• the amount of an acid to be used is 0 to 10, preferably 0.5 to 4, moles per mole of guanidino fatty acid amide of formula II.
• the reaction temperature is 0° to 100° C., usually from room temperature to 80° C., preferably 40° to 70° C.
• the reaction time varies with the reaction temperature. A reaction time of 1 to 2 days is preferred for increasing the yield.
• the ratio of guanidino fatty acid amide of formula II to dihydroxyethanamide of formula III is not specifically limited, it is general practice to use 0.5 to 4, preferably 0.8 to 1.5, moles of the latter for 1 mole of the former.
• the resulting compound is an N-[4-(3-aminopropyl)aminobutyl]-2-( ⁇ -guanidinofatty-acid-amido)-2-hydroxyethanamide represented by the general formula ##STR18## wherein Y and n are as defined above, which is the compound of formula I in which R is a hydrogen atom.
• the compounds of formula I in which R is the groups other than the hydrogen atom are obtained by alkylating the hydroxyl group at the position 11 of the compound of formula Ia with the aforesaid aliphatic alcohol, diazoparaffin or benzyl alcohol.
• the compound obtained by the above condensation or spergualin obtained from the microbial culture broth can be used for the compound of formula Ia.
• the compound obtained by the alkylation is an N-[4-(3-aminopropyl)aminobutyl]-2-( ⁇ -guanidinofatty-acid-amido)-2-alkoxyethanamide represented by the general formula ##STR19## wherein Y and n are as defined above and R' represents an alkyl group of 1 to 4 carbon atoms, which may have a hydroxyl group as substituent, or benzyl group.
• the alkylation is carried out in the following manner.
• R' is an alkyl group of 1 to 4 carbon atoms which may have a hydroxyl group as substituent, or a benzyl group.
• alcohols include lower alcohols such as methanol, ethanol, propanol and butanol, glycols such as ethylene glycol and propylene glycol, and benzyl alcohol.
• the reaction is carried out preferably in the alcohol of the above formula IV, though an inert solvent may be used.
• Suitable acid catalysts include inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as acetic acid and p-toluenesulfonic acid, and cationic exchange resins.
• the reaction temperature is in the range of from 0° to 100° C., generally from room temperature to 80° C., room temperature being most preferred.
• the reaction time varies with the reaction temperature and ranges from 1 hour to 10 days, preferably from 1 to 2 days.
• Suitable protective groups can be selected by referring to the literature (J. F. W. Mcomie, Ed., "Protective Groups in Organic Chemistry", Plenum Press, NY, 1973). Those protective groups for amino group which are generally used in the peptide synthesis are useful.
• Examples are monovalent protective groups such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl, trichloroethoxycarbonyl and isobornyloxycarbonyl and divalent protective groups such as phthaloyl and succinyl.
• monovalent protective groups such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl, trichloroethoxycarbonyl and isobornyloxycarbonyl and divalent protective groups such as phthaloyl and succinyl.
• aralkyloxycarbonyl groups such as benzyloxycarbonyl and p-methoxybenzyloxycarbonyl groups because of the ease of introduction and removal.
• the introduction of these protective groups is effected by known procedures and advantageously by the method of active este
• the amino and imino groups of the hydroxyethanamide of formula Ia are protected with the above-noted protective group and the resulting compound is subjected to the reaction with a diazoparaffin in an inert organic solvent such as methylene chloride or tetrahydrofuran at a temperature of -20° to 20° C., usually -10° to 10° C., preferably -3° to 3° C. for 1 to 15, usually 2 to 8, hours to effect alkylation.
• the reaction does not necessarily require a catalyst, but it is accelerated in the presence of a Lewis acid catalyst such as boron trifluoride, aluminum chloride, hydrofluoboric acid or selenium dioxide.
• diazoparaffins of 1 to 4 carbon atoms mention may be made of diazomethane, diazoethane, diazopropane and diazobutane. These diazoparaffins may be synthesized from corresponding N-nitrosoalkylurea, N-nitrosoalkylurethane, N-nitrosoalkylsulfonamide and N-nitrosoalkyl-N'-nitroguanidine by the known procedures [e.g.
• the alkylation with a diazoparaffin enables the hydroxyl group at the position 11 to become alkylated without change in the configuration at position 11 of the formula Ia.
• a (-) hydroxyethanamide of formula Ia is converted to the corresponding (-) alkoxyethanamide or an epimeric mixture ( ⁇ ) with respect to position 11 is obtained from the (+) hydroxyethanamide or ( ⁇ ) hydroxyethanamide, respectively.
• the hydroxyethanamide compound of formula Ia will have hydroxyl groups at both positions 11 and 15 when Y is ##STR20## In this case, owing to the difference in reactivity between two hydroxyl groups, it is possible to alkylate selectively the hydroxyl group at position 11.
• the protective groups for the amino and imino groups of the alkylated product can be removed in an ordinary way, leaving behind an alkoxyethanamide of formula Ib.
• the protective group is an aralkyloxycarbonyl group, it can be removed by the ordinary catalytic hydrogenation at atmospheric pressure.
• the reaction is carried out in a suitable solvent such as methanol, ethanol, dioxane or a mixture thereof in the presence of palladium or platinum as catalyst.
• the reaction is accelerated by the addition of an acid such as hydrochloric acid or acetic acid.
• the 2-substituted ethanamide of the formula I is N-[4-(3-aminopropyl)aminobutyl]-2-( ⁇ -guanidino- ⁇ -hydroxyfatty-acid-amido)-2-substituted-ethanamide represented by the general formula ##STR22## wherein R and n are as defined above, provided that when n is 4, R represents the groups other than the hydrogen atom.
• R and n are as defined above, provided that when n is 4, R represents the groups other than the hydrogen atom.
• HPLC high-performance liquid chromatography
• the compound of formula Ie is obtained by protecting the amino and imino groups of the compound of formula Id with the aforesaid protective groups, subjecting the resulting compound to dehydration treatment, and removing the protective groups.
• the dehydration can be effected, for example, by a known method in which dicyclohexylcarbodiimide is allowed to react in the presence of a copper (I or II) chloride [Journal of the American Chemical Society, 90, 3245 (1965)]. This method is preferable because it is carried out under mild neutral conditions. An excess of dicyclohexylcarbodiimide curtails the reaction time. In view of the solubility of starting materials, N,N-dimethylformamide is an example of suitable solvent.
• the reaction temperature is usually from room temperature to 100° C.
• the reaction time varies from several hours to several days depending upon the reaction temperature.
• the compound of formula If is advantageously obtained from the compound of formula Ie by the reduction of double bond.
• the reduction of double bond is effected by customary methods such as, for example, catalytic reduction. If the amino or imino protective group is an aralkyloxycarbonyl group, the reduction of double bond and the removal of protective groups are simultaneously achieved by the catalytic reduction.
• R' of the alkoxyethanamide compound of formula Ib is a benzyl group
• the benzyl group is removed by the catalytic reduction and the compound is converted to the hydroxyethanamide compound of formula Ia while retaining the configuration at position 11 unchanged.
• the catalytic reduction for the removal of a protecting aralkyloxycarbonyl group proceeds at a low rate under atmospheric pressure, whereas a satisfactory result is obtained with reduced reaction time by carrying out the reaction in aqueous acetic acid solution under a higher pressure of several to several tens atmospheres.
• ⁇ -guanidino fatty acid amide represented by the formula ##STR26## wherein Y and n are as defined above, which is a starting material for the synthesis of the compound of formula Ia, is synthesized as described below.
• This compound is an ⁇ -guanidino saturated fatty acid amide represented by the general formula ##STR27## wherein n is as defined above.
• ⁇ -guanidino saturated fatty acid amides are known compounds and can be synthesized from commercial raw materials by known reactions. For instance, the compound is obtained by protecting the amino group of an ⁇ -amino fatty acid of the formula
• n is as defined above, then esterifying the carboxyl group, treating the resulting ester with ammonia to convert into an amide, removing the amino protective group and converting the amino group to a guanidino group.
• the compound of formula IIa can also be prepared by oxidizing a diamine to a corresponding nitrile compound represented by the formula
• n is as defined above, then hydrolyzing the nitrile group to form an amide compound and converting the amino group to a guanidino group.
• ⁇ -guanidino saturated fatty acid amides of formula IIa include 4-guanidinobutanamide, 5-guanidinopentanamide, 6-guanidinohexanamide, 7-guanidinoheptanamide, 8-guanidinooctanamide, 9-guanidinononanamide, 10-guanidinodecanamide and 11-guanidinoundecanamide.
• This compound is an ⁇ -guanidino- ⁇ -hydroxy fatty acid amide represented by the general formula ##STR29## wherein n is as defined above, and is synthesized by known reactions in various ways. For instance, it is synthesized by protecting the amino group of an ⁇ -amino fatty acid represented by the formula
• n is as defined above, then extending the carbon chain by two carbon atoms, converting the resulting compound to a ⁇ -hydroxy fatty acid amide through a series of reactions generally used in preparing a ⁇ -hydroxycarboxylic acid derivative, then removing the protective group to regenerate the amino group, and converting the amino group into guanidino group.
• the procedure is further described below in detail with reference to an example.
• the ⁇ -guanidino- ⁇ -hydroxy fatty acid amide of formula IIb may be synthesized by protecting the amino group of an ⁇ -amino fatty acid of formula VII with an amino-protective group such as benzyloxycarbonyl group, converting the carboxylic acid into a reactive derivative such as an acid imidazolide, condensing the reactive derivative with magnesium enolate of monoethyl malonate of the formula ##STR30## [Buletin de la Societe Chimique de France, 945 (1964)] to yield a ⁇ -keto ester of the formula
• X' represents an amino-protective group and n is as defined above, reducing the ketone carbonyl group to form a ⁇ -hydroxy ester, treating the ester with ammonia to form an amide, removing the amino-protective group, and converting the regenerated amino group into guanidino group.
• ⁇ -guanidino- ⁇ -hydroxy fatty acid amides thus prepared, mention may be made of 4-guanidino-3-hydroxybutanamide, 5-guanidino-3-hydroxypentanamide, 6-guanidino-3-hydroxyhexanamide, 7-guanidino-3-hydroxyheptanamide, 8-guanidino-3-hydroxyoctanamide and 9-guanidino-3-hydroxynonanamide.
• (S)-7-guanidino-3-hydroxyheptanamide may be obtained by hydrolyzing with an acid or alkali the antibiotic substance BMG 162-aF2 (spergualin) represented by the formula ##STR31## which is isolated from the culture filtrate of a microorganism of the genus Bacillus, such as, for example, Bacillus BMG 162-aF2 (FERM-P 5230; ATCC 31932). It may also be synthesized, as described in the Journal of Antibiotics, Vol. 34, 1625 (1981) and in U.S. patent application Ser. No.
• This compound is an ⁇ -guanidino- ⁇ , ⁇ -unsaturated fatty acid amide represented by the formula ##STR32## wherein n is as defined above.
• These compounds are advantageously synthesized by the dehydration of ⁇ -guanidino- ⁇ -hydroxy fatty acid amides of formula IIb, the preparation of which is described above.
• the dehydration can be effected by the methods customarily used in dehydrating a ⁇ -hydroxy fatty acid amide, it is preferable to carry out the reaction under mild neutral conditions.
• One of the suitable ways is to effect dehydration by the action of dicyclohexylcarbodiimide in the presence of copper(II) chloride [Journal of the American Chemical Society, 90, 3245 (1968)].
• a preferable solvent is N,N-dimethylformamide because of the solubility of the acid addition salt.
• the reaction temperature is generally from room temperature to 100° C.
• the reaction time generally ranges from several hours to several days depending upon the reaction temperature. The reaction time can be reduced by using the dicyclohexylcarbodiimide in excess.
• Examples of ⁇ -guanidino- ⁇ , ⁇ -unsaturated fatty acid amides thus prepared include 4-guanidino-2-butenamide, 5-guanidino-2-pentenamide, 6-guanidino-2-hexenamide, 7 -guanidino-2-heptenamide, 8-guanidino-2-octenamide and 9-guanidino-2-nonenamide.
• the compound of formula III may also be obtained in a high yield by the hydrolysis of aforementioned antibiotic substance BMG 162-aF2 (spergualin).
• the collected effluent was further passed through a column (16 mm inner diameter) packed with 100 ml of Dowex® 50W-X4 (H-type).
• the column was washed successively with each 300 ml of water and 0.2M aqueous ammonia and eluted with 0.5M aqueous ammonia (10 ml fraction size).
• the fraction Nos. 16 to 33 were combined and evaporated to dryness to yield 2.73 g (48% yield) of a white powder of (S)-3-amino-7-benzyloxycarbonylaminoheptanoic acid (C 15 H 22 N 2 O 4 .H 2 O); decomposition point, 143°-147° C.; [ ⁇ ] D 22 +14° (c 1, methanol).
• the Amberlite® CG-50 column was eluted with 0.5N aqueous ammonia to recover 746 mg (24% recovery) of (S)-3,7-diaminoheptanoic acid.
• aqueous layer was evaporated to dryness, dissolved in 5 ml of water, passed through a column (20 mm inner diameter) packed with 50 ml of CM-Sephadex® C-25 (Na-type) and the column was eluted with 200 ml of 0.5M aqueous sodium chloride solution (10 ml fraction size).
• Fractions No. 17 to No. 30 were combined, evaporated to dryness and extracted 3 times with methanol.
• the methanol solution was passed through a column (20 mm inner diameter) packed with 150 ml of Sephadex® LH-20 and developed with methanol (5 ml fraction size).
• Fractions No. 9 to No. 16 were combined and evaporated to dryness.
• the aqueous layer was acidified with concentrated hydrochloric acid and extracted 3 times with 50 ml of ethyl acetate.
• the extract solutions were combined, washed with saturated saline, dried over anhydrous sodium sulfate and freed from the solvent by distillation to obtain 12.16 g (92% yield) of 6-benzyloxycarbonylaminohexanoic acid with a melting point of 127°-128° C.
• the reaction mixture was concentrated under reduced pressure and the resulting white powder was purified in a manner similar to that in Example 7, using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to yield 110 mg (60% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(6-guanidinohexanamido)-2-methoxyethanamide trihydrochloride.
• the aqueous solution was adjusted to pH 6 with 1N aqueous sodium hydroxide solution, then passed through a column (25 mm inner diameter) packed with 300 ml of CM-Sephadex® C-25 (Na-type) and fractionated by the gradient elution method with each 2 liters of water and 1M aqueous sodium chloride solution (fraction size, 17 ml).
• the fractions No. 138 to No. 152 corresponding to the sodium chloride concentrations of 0.59-0.65M were combined, evaporated to dryness and extracted twice with 10 ml of methanol.
• the reaction mixture was concentrated under reduced pressure and the resulting white powder was purified in a manner similar to that in Example 7, using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to yield 195 mg (86% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(8-guanidinooctanamido)-2-methoxyethanamide trihydrochloride.
• the reaction mixture was concentrated under reduced pressure and the resulting white powder was purified in a manner as that in Example 7, using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to yield 107 mg (65% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(9-guanidinononanamido)-2-methoxyethanamide trihydrochloride.
• the resulting white powder was purified in a manner similar to that in Example 7, using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20, to yield 71 mg (68% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(7-guanidinoheptanamido)-2-ethoxyethanamide trihydrochloride.
• reaction mixture was adjusted to pH 6 with 1N aqueous sodium hydroxide solution and purified in a manner similar to that in Example 7, using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20, to yield 63 mg (58% yield) of a white powder of N-(4-(3-aminopropyl)aminobutyl]-2-(7-guanidinoheptanamido)-2-(2-hydroxy)ethoxyethanamide trihydrochloride.
• aqueous layer was neutralized with Amberlite® IR-410, evaporated to dryness and purified in a manner similar to that in Example 7 using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to yield 61 mg (52% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(7-guanidinoheptanamido)-2-benzyloxyethanamide trihydrochloride.
• reaction mixture was purified in a manner similar to that in Example 1 using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to obtain 244.6 mg (46.5% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(7-guanidino-2-heptenamido)-2-hydroxyethanamide trihydrochloride.
• reaction mixture was purified in a manner similar to that in Example 1 using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to obtain 128.3 mg (29.2% yield) of N-[4-(3-aminopropyl)aminobutyl]-2-(8-guanidino-2-octenamido)-2-hydroxyethanamide trihydrochloride.
• reaction mixture was purified in a manner similar to that in Example 1 using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to obtain 135.0 mg (31.1% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(9-guanidino-2-nonenamido-2-hydroxyethanamide trihydrochloride.
• reaction mixture was purified in a manner similar to that in Example 1 using CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 to yield 120.7 mg (38.6% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(8-guanidino-3-hydroxyoctanamido)-2-hydroxyethanamide trihydrochloride.
• reaction mixture was purified in a manner similar to that in Example 1 using CM-Sephadex® C-25 (Na-type) and and Sephadex® LH-20 to yield 220.8 mg (33.4% yield) of a white powder of N-[4-(3-aminopropyl)aminobutyl]-2-(9-guanidino-3-hydroxynonanamido)-2-hydroxyethanamide trihydrochloride.
• reaction mixture was evaporated to dryness, then dissolved in 10 ml of water, adjusted to pH 4 with 1N aqueous sodium hydroxide solution and purified in essentially the same manner as in Example 22 using CM-Sephadex® C-25 (Na-type) and Sephadex® H-20, yielding 355.6 mg (70% yield) of a white powder of 11-O-ethylspergualin trihydrochloride.
• n-butanol soluble portion of the reaction mixture was evaporated to dryness, then dissolved in 10 ml of water, adjusted to pH 4 with 1N aqueous sodium hydroxide solution and purified with CM-Sephadex® C-25 (Na-type) and Sephadex® LH-20 in essentially the same manner as in Example 22 to obtain 114.7 mg (21% yield) of a white powder of 11-O-n-butylspergualin trihydrochloride.
• reaction mixture was concentrated under reduced pressure, dissolved in 10 ml of 0.5M aqueous sodium chloride solution, then passed through a column of Diaion®HP-20 (400 ml) equilibriated with 0.5M aqueous sodium chloride solution, washed with 1 liter of 0.5M saline, then with 1 liter of water and eluted with methanol (fraction size, 15 g).
• Fractions No. 21 to No. 30 were combined and evaporated to dryness to yield 287 mg (82% yield) of a white powder of (-)-1-N,4-bis(benzyloxycarbonyl)spergualin hydrochloride; [ ⁇ ] D 21 -11° (c 1, water).
• the diazomethane solution was prepared by gradually adding 10 g of N-nitrosomethylurea to a mixture of 30 ml of a 40% potassium hydroxide solution and 100 ml of methylene chloride while cooling at 40° C. in water; separating the organic layer and extracting the aqueous layer with 10 ml of methylene chloride; combining the organic layers and drying over granular potassium hydroxide at 5° C. for 3 hours.] After 3.5 hours from the start of reaction, stirring was discontinued.
• reaction mixture was concentrated under reduced pressure, then dissolved in 3 ml of 50% aqueous methanol, passed through a column of Diaion®HP-20 (100 ml), washed with 300 ml of 10% aqueous methanol and eluted with methanol (fraction size, 15 ml).
• Example 27(b) In a manner similar to that in Example 27(b), a methylene chloride solution of diazoethane was allowed to react with 352 mg (0.451 mmole) of the (-)-1-N,4-bis(benzyloxycarbonyl)spergualin hydrochloride obtained in Example 27(a) to yield 217.0 mg of a mixture of (-)-1-N,4-bis(benzyloxycarbonyl)-11-O-ethylspergualin hydrochloride and unreacted (-)-1-N,4-bis(benzyloxycarbonyl)spergualin hydrochloride.
• Example 27(c) The mixture was treated in essentially the same manner as in Example 27(c) to obtain 41.7 mg of a white powder of (-)-11-O-ethylspergualin trihydrochloride in an overall yield of 17.1%; [ ⁇ ] D 25 -24.8° (c 1, water).

Landscapes

• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Animal Behavior & Ethology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Medicinal Chemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=15695187

Family Applications (3)

Family Applications After (2)

Country Status (19)

Cited By (17)

Families Citing this family (12)

Citations (3)

Family Cites Families (4)

• 1981
  • 1981-10-08 JP JP56159503A patent/JPS5862152A/ja active Granted
• 1982
  • 1982-09-29 US US06/426,372 patent/US4518532A/en not_active Expired - Lifetime
  • 1982-10-04 DE DE19823236725 patent/DE3236725A1/de active Granted
  • 1982-10-05 AU AU89121/82A patent/AU554489B2/en not_active Expired
  • 1982-10-06 AT AT0369682A patent/AT393680B/de not_active IP Right Cessation
  • 1982-10-06 NL NL8203874A patent/NL193107C/nl not_active IP Right Cessation
  • 1982-10-06 SE SE8205698A patent/SE452006B/sv not_active IP Right Cessation
  • 1982-10-06 ES ES516258A patent/ES516258A0/es active Granted
  • 1982-10-06 CA CA000412980A patent/CA1187513A/fr not_active Expired
  • 1982-10-07 HU HU823215A patent/HU187417B/hu unknown
  • 1982-10-07 KR KR8204571A patent/KR870000656B1/ko active
  • 1982-10-07 IT IT49228/82A patent/IT1189373B/it active
  • 1982-10-07 CH CH5895/82A patent/CH653015A5/de not_active IP Right Cessation
  • 1982-10-07 GB GB08228636A patent/GB2111480B/en not_active Expired
  • 1982-10-07 DK DK445182A patent/DK166080C/da active
  • 1982-10-07 CS CS827179A patent/CS268654B2/cs unknown
  • 1982-10-08 FR FR8216894A patent/FR2514350B1/fr not_active Expired
  • 1982-10-08 BE BE0/209206A patent/BE894651A/fr unknown
  • 1982-10-08 YU YU2276/82A patent/YU44678B/xx unknown
• 1983
  • 1983-10-14 ES ES526501A patent/ES526501A0/es active Granted
• 1985
  • 1985-04-15 US US06/723,169 patent/US4658058A/en not_active Expired - Lifetime
• 1988
  • 1988-03-15 US US07/170,611 patent/US4983328A/en not_active Expired - Lifetime

Patent Citations (4)

Non-Patent Citations (12)

Also Published As

Similar Documents

Legal Events